This invention relates to the application of recombinant DNA technology in the field of neurobiology. More particularly, the invention relates to the cloning and expression of DNA coding for proteins which modulate the function of glutamate receptors.
In the mammalian central nervous system (CNS), the transmission of nerve impulses is controlled by the interaction between a neurotransmitter substance released by the xe2x80x9csendingxe2x80x9d neuron which then binds to a surface receptor on the xe2x80x9creceivingxe2x80x9d neuron, to cause excitation thereof. L-glutamate is the most abundant neurotransmitter in the CNS, and mediates the major excitatory pathway in vertebrates. Glutamate is therefore referred to as an excitatory amino acid (EAA) and the receptors which respond to it are variously referred to as glutamate receptors, or more commonly as EAA receptors.
Members of the EAA receptor family can be grouped into three main types based on differential binding to certain glutamate analogs. One type of EAA receptor, which in addition to glutamate also binds the compound NMDA (N-methyl-D-aspartate), is referred to as the NMDA type of EAA receptor. Two other glutamate-binding types of EAA receptor, which do not bind NMDA, are named according to their preference for binding with two other EAA receptor agonists, namely AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate), and kainate (2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetate). Accordingly, receptors which bind glutamate but not NMDA, and which bind with greater affinity to kainate than to AMPA, are referred to as kainate-type EAA receptors. Similarly, those EAA receptors which bind glutamate but not NMDA, and which bind AMPA with greater affinity than kainate are referred to as AMPA-type EAA receptors.
The glutamate-binding EAA receptor family is of great physiological and medical importance. Glutamate is involved in many aspects of long-term potentiation (learning and memory), in the development of synaptic plasticity, in epileptic seizures, in neuronal damage caused by ischemia following stroke or other hypoxic events, as well as in other forms of neurodegenerative processes. The development of therapeutics which modulate these processes is being slowed by the lack of any homogeneous source of receptor material with which to discover selectively binding drug molecules, which interact specifically at the interface of an appropriate EAA receptor. The brain derived tissues currently used to screen candidate drugs are heterogeneous receptor sources, possessing on their surface many receptor types which interfere with studies of the EAA receptor/ligand interface of interest. The search for human therapeutics is further complicated by the limited availability of brain tissue of human origin. It would therefore be desirable to obtain cells that are genetically engineered to produce only the receptor of interest. With cell lines expressing cloned receptor cDNA, a substrate which is homogeneous for the desired receptor is provided, for drug screening programs.
Non-human cDNAs which appear to encode the NMDA-type of EAA receptor have recently been identified and isolated. A cDNA encoding a subunit polypeptide of an NMDA receptor in rat, designated NR1, has been isolated as described by Moriyoshi et al. in Nature 354: 31, 1991. An extension of this work has revealed seven isoforms of NR1, presumably generated by combinations of alternative RNA splicing in the amino- and carboxy-terminal regions of NR1 (Anantharam et al. FEBS Lett. 305: 27, 1992; Durand et al. Proc. Nati. Acad. Sci. USA 89: 9359, 1992; Nakanishi et al. Proc. Natl. Acad. Sci. USA 89: 8552, 1992; Sugihara et al. Biochem, Biophys. Res. Commun. 185; 826, 1992; Hollmann et al. Neuron 10; 943, 1993; Kusiak and Norton. Mol. Brain. Res. 20: 64, 1993). DNA encoding NR1 and one of its isoforms have also been cloned from mouse brain by Yamazaki et at. as described in FEBS Lett. 300: 39, 1992. Other rat NMDA receptor subunits, designated NR2A, NR2B, NR2C and NR2D, have also been identified (Monyer et al. Science 256: 1217, 1992; Ishii et al. J. Biol. Chem. 268: 2836, 1993), as well as mouse NMDA receptor subunits which have been designated xcex51, xcex52, xcex53 and xcex54 (Meguro et al. Nature 357: 70, 1992; Kutsuwada et al. Nature 358: 36, 1992; Ikeda et al. FEBS Lett. 313: 34, 1992).
There has emerged from these molecular cloning advances, a better understanding of the structural features of NMDA receptors and their subunits, as they exist in the non-human brain. According to the current model, each NMDA receptor is heteromeric, consisting of individual membrane-anchored subunits, each comprising transmembrane regions and extracellular domains that dictate ligand-binding properties and contribute to the ion-gating function served by the receptor complex.
In the search for therapeutics useful to treat CNS disorders in humans, it is highly desirable to obtain knowledge of human EAA receptors, and proteins which modulate the activity of these receptors. Such an understanding would provide a means to screen for compounds that selectively interact with this activity, i.e. to stimulate or inhibit receptor activity, thereby providing a means to identify compounds having potential therapeutic utility in humans. Non-human mammalian models are not suitable for this purpose despite significant protein homology due to the fact that minute sequence discrepancies have been found to cause dramatic pharmacological and functional variation between species homologues of the same protein (Oksenberg et al., Nature, 360:161, 1992; Hall et al. Trends Pharmacol. Sci. 14: 376, 1993). It is therefore particularly desirable to provide cloned cDNA encoding human EAA receptor proteins or modulatory proteins thereof, and cell lines expressing these proteins, in order to generate a screening method for a, compounds therapeutically useful in humans. These, accordingly, are objects of the present invention.
Human cDNAs encoding NMDA receptor modulatory proteins have been identified and characterized, and include proteins referred to herein as the NR3 and NR4 modulatory proteins. Specifically encompassed are parent proteins designated the NR3-1 and NR4-1 proteins, as well as functional sequence-related variants of NR3-1 and NR4-1, and functional fragments of NR3-1 and NR4-1.
In one of its aspects, thus, the present invention provides an isolated polynucleotide, consisting either of DNA or of RNA, which codes for a human NR3 protein, or functional fragments thereof.
In another aspect of the present invention, there is provided a cell that has been genetically engineered to produce a human EAA receptor modulatory protein belonging to the herein-defined NRS family. In related aspects of the present invention, there are provided recombinant DNA constructs and methods useful to obtain substantially homogeneous sources of the human NR3 protein, comprising the steps of culturing the genetically engineered cells, and then recovering the cultured cells.
In another aspect of the present invention, there is provided a method for evaluating interaction between a candidate ligand and a human EAA receptor modulatory protein, which comprises the steps of incubating the candidate ligand with a genetically engineered cell as described above, or with a membrane preparation derived therefrom, and then assessing said interaction by determining the extent of protein/ligand binding, or by determining the ligand-induced electrical current across said cell.
In yet another aspect of the present invention, a cell that has been engineered genetically to produce a human heteromeric NR3/receptor complex comprising an NR3 protein and an NMDA receptor is provided.
In a further aspect of the present invention, there is provided a method for evaluating interaction between a candidate ligand and a human heteromeric NR3/receptor complex comprising an NR3 protein and an NMDA receptor, said method comprising the steps of incubating the candidate ligand with a cell line engineered to produce said receptor complex, or with a membrane preparation derived therefrom, and then assessing the interaction therebetween by determining the extent of protein/ligand binding, or by determining the ligand-induced electrical current across said cell.
Other aspects of the present invention include a human NR3 protein, in a form essentially free from other proteins of human origin, functional and immunogenic fragments of the protein, antibodies which bind to the protein, and oligonucleotides which hybridize to nucleic acid encoding the protein.
Other aspects of the present invention, which encompass various applications of the discoveries herein described, will become apparent from the following detailed description, and from the accompanying drawings in which: